PROJECT SUMMARY Novel T-cell immunotherapies that infuse multi-virus-specific, cytotoxic T-cells to boost the host immune response against viruses may be a promising treatment option for persistent viral infections in transplant recipients. However, causes of T-cell therapy failures in early clinical trials are not yet fully understood and virus-specific causes for treatment failures are currently not known. With advances in next-generation sequencing, whole genome sequencing of the most clinically relevant viruses in transplantation for virus escape mutant detection is now possible, though methodologies and informatics pipelines have yet to be established. The objective of this proposal is to develop an efficient sequencing method that provides genome-wide data on five of the most common viruses targeted by multi-virus cytotoxic T-cell therapies: adenovirus (ADV), BK virus (BKV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus-6 (HHV-6) to facilitate the detection of virus escape mutants as a potential cause of T-cell therapy failure and define virus genetic changes in response to immune pressure. The central hypothesis is that therapeutic strategies that enhance the host immune response against viruses, such as the infusion of virus-specific cytotoxic T-cells, will promote the emergence of escape mutants and result in treatment failure and persistent viremia. We plan to test the central hypothesis and accomplish the objectives of this proposal by pursuing these specific aims: 1) Develop and validate a whole genome sequencing platform for ADV, CMV, EBV, and HHV-6; and 2) Define virus responses to T-cell therapy in transplant recipients with persistent viremia. Under the first aim, a novel design that uses bait enrichment for targeted virus DNA sequencing will be used and informatics pipeline validated, to accompany BKV whole genome sequencing capabilities recently established by our group. Under the second aim, the platform will be used to define changes in the virus repertoire in transplant recipients with recurrent or persistent viremia after receipt of virus-specific T-cell immunotherapy. The significance of this work is needed application to emerging clinical trials evaluating the efficacy of novel immune-based therapies in transplant recipients at institutions worldwide. The proposed research is innovative, in the applicant's opinion, because it enables whole genome sequencing of five of the most important viruses causing disease in transplant recipients, not yet available, that can be easily and broadly applied for characterization of these viruses in transplantation and other immunocompromised states. Such depth of virus characterization offers the ability to evaluate dynamic changes in the virus repertoire to both immunomodulating and antiviral therapies in various disease states. Thus, the proposed work is expected to open new research horizons not previously attainable and improve the management of these pervasive viral infections.